Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase make up the family of pterin-dependent aromatic amino acid hydroxylases. These non-heme iron monooxygenases catalyze key steps in the metabolism of higher eukaryotes: catabolism of excess phenylalanine, catecholamine biosynthesis, and serotonin biosynthesis, respectively. They contain homologous catalytic domains of about 300 amino acid residues which contain all the residues responsible for catalysis and substrate specificity. Mechanistic and structural studies to date have allowed the proposal of a unified chemical mechanism for these three enzymes, while structural studies are beginning to identify amino acid residues critical for catalysis and substrate specificity. In the next grant period the focus will continue to be on tyrosine hydroxylase, with selected experiments designed to identify common features and differences among the three enzymes. Experiments are proposed to further investigate individual steps in the proposed catalytic mechanism, utilizing a variety of kinetic and spectroscopic approaches. These are combined with structural approaches which address the roles of individual amino acid residues in catalysis and the timing of conformational changes which occur during the catalytic cycle. The results will be of importance in understanding the properties of these three physiologically critical enzymes and the general strategies by which biological systems carry out hydroxylation reactions.